LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

OBJECTIVE: In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times. METHODS: 3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 μg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed. RESULTS: Cells activated by increasing doses of LPS upregulated TNF-α expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 °C. The chemokines MIP-1α/CCL3 and MIP-1β/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments. CONCLUSION: These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.